Inhibition of Lipopolysaccharide E. coli-induced acute lung injury by extracted Antidesma bunius (L.) Spreng fruits as compared to Fluticasone Propionate, a corticosteroid

The hallmark of Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is inflammation-induced alveolar-vascular barrier destruction and neutrophilic infiltration that leads to the formation of cytokines and oxygen radicals. The objective of the study is to investigate the protective and toxicological effects of Antidesma bunius (L.) Spreng [Bignay] in murine model of Lipopolysaccharide E. coli (LPS)-induced ALI and compared with Fluticasone Propionate (FP), a synthetic corticosteroid. We showed that extracted Bignay fruits have high amount of phenols, steroids and flavonoids but insignificant amount of heavy metals and aflatoxins. BALB/c mice of either sex were divided into 4 groups in the ALI mouse model; Group 1: vehicle control; Group 2: LPS alone; Group 3: Bignay + LPS; and Group 4: FP + LPS. Bignay and FP were administered via intraperitoneal injection while LPS was given intra-tracheally. Biomarkers of ALI such as total lung inflammatory cell count, total lung protein content, lung edema and interleukin-6 (IL-6) secretion were measured 24 hrs after vehicle control or LPS treatment. Compared to vehicle controls, LPS caused significant increased in all measured biomarkers of ALI in samples collected from bronchoalveolar lavage fluid and were significantly attenuated by Bignay fruit extract or FP. Pulmonary vascular leakage caused by LPS was also evaluated after injection with Evans blue dye, an indication of lung injury. Extracted Bignay fruits or FP when given to mice 2 hrs after LPS administration substantially decreased the pulmonary vascular leak. Our findings are the first evidence demonstrating the preventive and non-toxic effects of extracted Bignay fruits in a murine model of LPS-induced ALI. The results could be attributed to the presence of active secondary metabolites such as flavonoids, phenols and steroids. It is also evident that extracted Bignay fruits are as effective as FP, well-established steroid, in blocking the biomarkers of ALI caused by LPS.

183 method [39]. The mixture of Bignay (1 mL sample + 4 mL water) was placed in 50 184 mL volumetric flask. A 0.3 mL 5% NaNO 2 was added to the mixture and was kept 185 in dark place for 6 min; thereafter, 0.3 mL 10% AlCl 3 was added. Five min later, 5% 186 NaOH was added to complete the reaction. Absorbance of sample against blank 187 was read at 510 nm using Spectrophotometer (BMG Lab Tech, Germany).   The total lung protein was measured by Bradford assay (Fig 3). 378 substantial secretion of IL-6 as compared to baseline value (Fig 4). Extracted

Airway inflammatory cell lung infiltration
405 LPS caused inflammatory cell infiltration in the lung interstitium, surrounding 406 the airways and pulmonary blood vessels (Fig 5B) compared to vehicle control 407 treated mice (Fig 5A). A marked reduction of migrated cells to the site of 408 inflammation caused by LPS stimulation was reduced by 1000 mg.kg -1 Bignay (Fig   409 5C). Bignay was as effective as FP in reduction of cell infiltrate (Fig 5D) 410 suggesting that Bignay could be a good candidate in protecting the migration of 411 inflammatory cells to the site of inflammation.

452
We first analyzed the amount of heavy metals (   [50].

LPS administration increased the number of inflammatory cells migrating
499 into the lungs (Fig 2) and the overall BALF protein content of the lungs (Fig 3). In  (Fig 7).

545
The 561 In addition, the isolation or structural formula of active compounds in Bignay was 562 not included in this analysis, but biochemical compound recognition plus the anti-563 inflammatory activity of the three secondary metabolites were quantified. Although 564 the content of total aflatoxins and aflatoxin B1 were determined, the source of 565 Aspergillus Flavus and Apergillus parasiticus were not identified in this study.
566 Histological airway analysis is close to that of humans with ALI; our data in mice, 567 however, cannot be explicitly extrapolated to the human state.

568
As for ALI in humans, therapy does not confer therapeutic effectiveness 569 after significant histological changes occur. Although the blocking effects on ALI 570 production in this study are relatively short, a hyper-acute model of ALI is the 30 572 humans, ALI typically grows over a considerably longer period (hrs to days), as in a 573 cumulative rather than square wave fashion, the inciting stimulus likely causes lung 574 injury. Therefore, the demonstration that Bignay administered pre-LPS is 575 successful in abolishing or altering ALI in mice for up to 2 hrs indicates the 576 likelihood of a longer window for intervention in the more chronic human production 577 of ALI. This depends, of course, on the degree to which the results in this study 578 translate into the human situation. It is also presumed that until ALI is fully 579 manifested, this therapy is probably helpful.

580
We also observed that complete blockade of ALI caused by Bignay is not all 581 done in treated mice. Nonetheless, with 1000 mg.kg-1 Bignay, a significant or near 582 complete reduction of the impact of LPS on ALI can be induced, especially in the 583 reduction of vascular leakage caused by LPS stimulation (Fig 6). This blocking 584 effect is as efficient as FP in LPS-induced ALI/ARDS in mice, indicating that Bignay